Most procedures in molecular genetics require means for introducing nucleic acids into cells. This is usually accomplished by chemical transformation (e.g., CaCl.sub.2 treatment), electroporation or, for E. coli, in vitro packaging of phage lambda. All of these methods are somewhat labor-intensive and time consuming, particularly, if a procedure requires many cycles of isolating, manipulating and transforming DNA. Furthermore, the efficiency of the procedures is relatively low. For example, even when transforming purified supercoiled DNA, at best, about 1/100 molecules become stably established in a cell. For ligation mixtures, the efficiencies are 2-3 orders of magnitude lower. Even these efficiencies are applicable to only a relatively small number of preferred cell types commonly used in genetic engineering. It would be desirable to be able to obtain high transfection efficiency in any cell of interest.
A few bacterial isolates are naturally competent (i.e., are capable of taking up DNA from their medium). Reports exist for Bacillus, Neisseria (Rudel et al., PNAS 92, 7986-7990 (1995); Facius & Meyer, Mol. Microbiol. 10, 699-712 (1993)); Haemophilus (Williams et al., J. Bacteriol. 176, 6789-6794 (1994)), Helicobacter (Haas et al., Mol. Microbiol. 8, 753-760 (1993)), Acinetobacter (Lorenz et al., Arch. Microbiol., 157, 355-360 (1992)), Streptococcus (Lopez et al., J. Gen. Microbiol. 135, 2189-2197 (1989)), Campylobacter (Nedenskov-Sorensen, J. Infect. Dis. 161, 356-366 (1990)), Synechocystis (Barten & Lill, FEMS Microbiol. Lett. 129, 83-88 (1995)), Lactobacillus and Amycolatopis (Vrijbloed et al., Plasmid 34, 96-104 (1995)).
Some information has emerged concerning the genetic basis of natural competence in bacteria. Some genes have been identified and correlated with a role in mediating DNA uptake. In Neisseria, two proteins, PilC and PilE, having roles in phase variation, have been shown to be essential for natural competence (Rudel et al., Proc. Natl. Acad. Sci. USA 92, 7986-7990 (1995)). PilE is the major pilus subunit protein, and PilC functions in assembly and adherence of gonococcal pili. Both genes serve to convert linearized plasmid DNA into a DNase-resistant form. DNA uptake requires a Neisseria-specific uptake signal on the DNA and a functional RecA protein. DNA is taken up in linear form. Transformation with non-episomal DNA fragments requires homology to the chromosomal DNA to allow integration by homologous recombination. Other genes required for DNA uptake, called dud, and for transformation uptake, called ntr, have been identified (Biswas et al., J. Bact., 171, 657-664 (1989)). In Haemophilus, the sxy gene has been reported to be essential for competence. Overexpression of the sxy gene product confers constitutive competence on wildtype Haemophilus cells, (Williams et al., J. Bact., 176, 6789-6794 (1994)). In E. coli, the comA gene has been reported to be involved in natural competence (Facius & Meyer, Mol. Microbiol., 10, 699-712 (1993)). Regulatory genes involved in competence are the homologs of the E. coli cya gene, encoding adenylate cyclase, and E. coli crp genes, encoding the cAMP receptor protein.
The present invention is generally directed to transferring genes conferring DNA-uptake capacity in one species to another and evolving the genes so that they also confer comparable or better DNA-uptake capacity in the second species and/or the original species. Genes are evolved by a process termed recursive sequence recombination which entails performing iterative cycles of recombination and screening/selection. Cells expressing the evolved genes can be transfected without undertaking the time consuming preparatory steps of prior methods and/or with greater efficiency than the cells of prior methods.